Methods of non-destructive nanostraw intracellular sampling for longitudinal cell monitoring

ABSTRACT

Methods and apparatuses to non-destructively and periodically sample a small quantity of intracellular proteins and mRNA from the same single cell or cells for an extended period of time. Specifically, describe herein are non-perturbative methods for time-resolved, longitudinal extraction and quantitative measurement of intracellular proteins and nucleic acids from a variety of cell types using systems including nanostraws.

CROSS REFERENCE TO RELATED APPLICATIONS

This patent application is a continuation of U.S. patent applicationSer. No. 16/332,684, filed on Mar. 12, 2019, titled “METHODS OFNON-DESTRUCTIVE NANOSTRAW INTRACELLULAR SAMPLING FOR LONGITUDINAL CELLMONITORING,” now U.S. Patent Application Publication No. 2019/0359974,which is a national phase application under 35 USC 371 of InternationalPatent Application No. PCT/US2017/051392, filed Sep. 13, 2017, titled“METHODS OF NON-DESTRUCTIVE NANOSTRAW INTRACELLULAR SAMPLING FORLONGITUDINAL CELL MONITORING,” now International Publication No.2018/053020, which claims priority to U.S. Provisional PatentApplication No. 62/394,089, filed Sep. 13, 2016, and titled “METHODS OFNON-DESTRUCTIVE NANOSTRAW INTRACELLULAR SAMPLING FOR LONGITUDINAL CELLMONITORING”. Each of these patent applications is herein incorporated byreference in its entirety.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under contract HL133272awarded by the National Institutes of Health and under contract70NANB15H268 awarded by the National Institute of Standards andTechnology. The Government has certain rights in the invention.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference in their entirety to the sameextent as if each individual publication or patent application wasspecifically and individually indicated to be incorporated by reference.

FIELD

The present invention relates generally to methods for accessing andsampling from intracellular spaces, in particular methods for accessingand sampling from intracellular spaces using nanostraw systems.

BACKGROUND

Quantitative analyses of intracellular components, such as proteins andmRNA, may provide crucial information to decipher cellular behaviorrelated to disease pathogenesis, cellular senescence, development anddifferentiation. Increasingly sensitive, and even single-cell, mRNA andprotein detection methods have been developed, leading to new insightsinto cell function, phenotype heterogeneity, and noise in cellularsystems. Although powerful, these methods are hampered by the need tolyse the cell to extract the intracellular contents, providing only asingle snapshot in time without information about prior or futurestates. This is particularly problematic when studying dynamictransformations, including induced pluripotency and differentiation, orstochastic noise in gene expression at the single cell level. Phenotypeheterogeneity and fluctuations in single cells imply that cells inparallel cultures are often not representative, highlighting the needfor non-destructive sampling from the same set of cells repeatedly overtime.

Although time-resolved, longitudinal monitoring of some cells (e.g.,sampling the same population of cells periodically) has been possible tosome extent with intracellular fluorescence techniques, these techniquestypically do not allow ongoing sampling of otherwise unmarked cellularcomponents. Such techniques typically require genetically encodedfluorescent protein (FP)-based biosensors to nondestructively followintracellular enzymatic activity. In addition, it has monitoring of twoto five species of proteins in living cells has been demonstrated usingfluorescence resonance energy transfer (FRET) biosensors and bimolecularfluorescence complementation (BiFC), however, the number ofintracellular targets is still limited due to spectral overlap. Thepresence of the FP label may also interfere with the function of thefused protein, and validating the specificity of the sensor is crucial.Further, the transfection of the FP gene is itself an intrusive process.Genetically encoded biosensors, such as quantum dot (QD) labeledantibodies and molecular beacons, are also used for intracellulardetection, yet are challenging to deliver intracellularly, andperturbation of the cell due to the labeling methods and presence oflabel is still a significant concern. Overall, even with theavailability of FP methods, longitudinal studies are relatively rare.

Nanotechnology provides an alternative approach by taking advantage ofnanoscale dimensions to non-destructively introduce sensors into cells,or to extract small quantities of cellular contents. For example, ananowire ‘sandwich assay’ has been proposed, in which ˜100 nm diameternanowires functionalized with antibodies penetrate the cell with limitedtoxicity to bind specific enzymes for extraction. Actis et al. (Actis,et al., Compartmental Genomics in Living Cells Revealed by Single-CellNanobiopsy, ACS Nano. 2014 Jan. 28; 8(1):546-553) demonstrated a‘nanobiopsy’ which extracts fluid (e.g., approximated to be 50 fL) fromthe cytoplasm a single cell without cell cytotoxicity with around 70%success rate. Another nano-sampling approach have also been describedusing an AFM-based sampling platform with controlled pL volumeextraction, followed by a single time-point mRNA analysis. Thistechnique was found to be largely non-destructive, with 86% cellviability, demonstrating that cells may lose a fraction of their volumewithout apoptosis. Intracellular protein sampling was also possibleusing magnetized carbon nanotubes coated with poly-1-tyrosine to extractgreen fluorescent protein (GFP) from a cell culture, with better than70% cell viability. These promising results indicate that insertion andsampling at a single time-point is possible. However, none of theseapproaches repeatedly sampled from the same set of cells to follow theirexpression over time, nor provided quantitative assessment of themeasured quantities compared with the actual intracellular contents.Described herein are methods and apparatuses that may address the needsand problems mentioned above.

SUMMARY OF THE DISCLOSURE

Described herein are methods and apparatuses to non-destructively andperiodically sample a small quantity of intracellular proteins and mRNAfrom the same single cell or cells for an extended period of time.Specifically, describe herein are non-perturbative methods fortime-resolved, longitudinal extraction and quantitative measurement ofintracellular proteins and nucleic acids from a variety of cell typesusing systems including nanostraws.

Here we report a non-perturbative method for time-resolved, longitudinalextraction and quantitative measurement of intracellular proteins andmRNA from a variety of cell types. Using these methods and apparatuses,cytosolic contents were repeatedly sampled from the same cell orpopulation of cells for over 5 days through a cell culture substrateincorporating hollow nanostraws having an inner diameter of, e.g.,between 20 and 1000 nm (e.g., between 20-900 nm, between 20-800 nm,between 20-700 nm, between 20-600 nm, between 20-500, between 20-400,between 20-300, between 20-200 nm, etc.) and an outer diameter ofbetween, e.g., 50 and 1500 nm (e.g., between 50-1400 nm, between50-1300, between 50-1200 nm, between 50-1100 nm, between 50-1000 nm,between 50-900 nm, between 50-800 nm, between 50-700 nm, between 50-600nm, between 50-500 nm, between 50-400 nm, between 50-300 nm, between50-200 nm, etc.) within a defined sampling region. The techniques andapparatuses described herein may open, for a discrete time period, poresor gaps within the portion of the cell membrane over the contents may beextracted by a highly focused electroporation technique at the nanostrawdistal opening in contact with the cell membrane, allowing diffusion ofintracellular components (which may be driven by an applied electricalfield) for sampling from the nanostraw.

Once extracted, the cellular contents may be analyzed with conventionalmethods, including fluorescence, enzymatic assays (ELISA), andquantitative real-time polymerase chain reaction (qPCR). This process isnon-destructive, with >95% cell viability after sampling, enablinglong-term analysis. Importantly, the measured quantities from the cellextract have been found to constitute a statistically significantrepresentation of the actual contents within the cells. For example, aswill be described herein, of 48 mRNA sequences analyzed from apopulation of human cardiomyocytes derived from pluripotent stem cells(hiPSC-CMs), 41 were accurately quantified. The methods and apparatusesdescribed herein may sample from a select sub-population of cells withina larger culture, allowing native cell-to-cell contact and communicationeven during vigorous activity such as cardiomyocyte beating. Thesemethods and apparatuses may be applied to both cell lines and primarycells (including, but not limited to the examples provided herein, e.g.,Chinese hamster ovary cells, hiPSC-CMs, and human astrocytes derived in3D cortical spheroids). By tracking the same cell or group of cells overtime, these methods and apparatuses offer new avenues to understanddynamic cell behavior, including processes such as induced pluripotencyand differentiation.

For example, described herein are methods of nondestructive sampling ofintracellular sample material from within a cell. These methods mayinclude sampling at a single time point or at multiple time points. Forexample, any of these methods may include: applying a voltage between anupper electrode and a lower electrode through a nanostraw to open one ormore pores in a portion of the cell membrane extending over an openingof the nanostraw; capturing a sample material released from within thecell and into the nanostraw in a sample collector beneath the nanostraw;and stopping the application of voltage between the upper and lowerelectrodes and allowing the cell membrane to recover before more than15% of the sample material within the cell is released.

Applying a voltage between an upper electrode and a lower electrodethrough a nanostraw to open one or more pores in a portion of the cellmembrane extending over an opening of the nanostraw may include applyinga pulsed (e.g., positive, negative, and/or biphasic) voltage pulses, orcurrent pulses. The pulses may have a fixed or varying pulse width andpulse rate. The voltage may be applied for any appropriate duration. Thevoltage parameters, including the voltage duration, may be set by thecell size and type, and may be determined empirically, to prevent celldeath. As described in greater detail herein, the applied voltage(including the pulse parameters) may be selected to prevent release, bydiffusion and/or charge driven (e.g., electrophoresis) mobility. Forexample, applying may comprise applying a pulsed voltage of between 1and 100 V between the upper electrode and the lower electrode through ananostraw, e.g., having a pulse width of between about 10 microseconds(μs) and 50 milliseconds (ms) (e.g., such as between 10 μs and 10 ms,between 20 μs and 5 ms, between 20 μs and 1 ms, between 20 μs and 500μs, etc.). The voltage may be applied for a duration that allows samplematerial to move out of the cell, through the temporary cell membraneopening and into the nanostraw, so that it may be captured by the samplecollector.

Typically, the application of voltage to form openings in the cellmembrane is stopped before more than 25% (e.g., more than 20%, more than15%, more than 14%, more than 13%, more than 12%, more than 11%, morethan 10%, more than 9%, more than 8%, more than 7%, more than 6%, morethan 5%, etc.) of the sample material within the cell is released.Beyond this point, the cell may be more likely to die, and therefore itis beneficial to stop the application of voltage (and to allow the cellto recover). For example, the application of voltage may be stoppedbefore more than 15% of the sample material within the cell is releasedthrough the opening(s) in the cell membrane. The amount of samplematerial released through the cell may be dependent on the strength ofthe applied electrical field (e.g., the applied voltage), the cell size,and the size (e.g., diameter) of the nanostraw. The more charged aparticular type of sample material is, the more quickly it will bereleased from within the cell, dependent on the size of the samplematerial and the applied electrical field. Further, the larger thediameter (e.g., opening size) of the nanostraw, the more sample materialthat may be released. Typically, stopping the application of voltagebetween the upper and lower electrodes and allowing the cell membrane torecover before more than 15% of the material within the cell is releasedmay include stopping the application of a train of pluses of between 1and 100 V having a pulse width of between about 10 microseconds and 50milliseconds after a between 1 second and 300 seconds.

The sample may be captured (e.g., collected) in the sample collector inany appropriate manner For example, the sample material may remainsuspending in a fluid sample, and/or it may be immobilized on asubstrate, including bound or captured to a substrate. For example,capturing the sample may comprise immobilizing the sample material ontoa capture substrate.

In general, a plurality of nanostraws may be used, including a pluralitywithin each of a plurality of sample regions, and/or plurality acrossmultiple sample regions. For example, applying the voltage may compriseapplying the voltage between an upper electrode and a lower electrodethrough the nanostraw and a plurality of additional nanostraws within asample region. Further, capturing the sample may comprises capturing thesample material released into the nanostraw and the plurality ofadditional nanostraws in the sample collector. The nanostraws may beidentical or different.

The steps described above (e.g., applying voltage, capturing samplematerial, etc.) may be repeated, for the same cell or cells, over time.For example, any of these methods may include repeating, after a minimumrecover time, the steps of reapplying the voltage between the upper andlower electrode through the nanostraw, capturing sample material, andstopping the application of voltage, wherein the minimum recovery timeis longer than one hour. Typically, the minimum recovery time may belonger than a few minutes (e.g., longer than 10 minutes, longer than 15minutes, longer than 20 minutes, longer than 30 minutes, longer than 45minutes, longer than 1 hour, longer than 1.5 hours, longer than 2 hours,longer than 3 hours, longer than 4 hours, longer than 5 hours, longerthan 6 hours, longer than 12 hours, longer than 20 hours, etc.)

The method of claim 1, further comprising saving a first time samplefrom the captured sample material and repeating, for a plurality ofadditional repetitions after a minimum recovery time between eachrepetition, the steps of: reapplying the voltage between the upper andlower electrode through the nanostraw, capturing sample material, andstopping the application of voltage, wherein an additional time sampleis saved from the captured sample material for each repetition.

A sample material may include a single type or species of material(e.g., a particular protein, mRNA, etc., which may be generally referredto herein as a biomarker) or the sample material may be a mixture of avariety of different sample materials. Any of these methods may includedetecting the captured sample material captured in the sample collector.For example, any of these methods may include quantifying the capturedsample material captured in the sample collector. Any of these methodsmay include identifying a plurality of different biomarkers from thecaptured sample material. For example, any of these methods may includequantifying a plurality of different biomarkers from the captured samplematerial.

A method of nondestructive sampling of intracellular sample materialfrom within a cell at multiple time points, may include: applying avoltage of between 1 and 100 V between an upper electrode and a lowerelectrode through a nanostraw to open one or more pores in a portion ofthe cell membrane extending over an opening of the nanostraw; capturinga sample material released from within the cell and into the nanostrawin a sample collector beneath the nanostraw, wherein capturing comprisesimmobilizing the sample material onto a capture substrate; stopping theapplication of voltage between the upper and lower electrodes andallowing the cell membrane to recover before more than 15% of the samplematerial within the cell is released; and allowing the cell to recoverfor a minimum recovery time of at least 1 hour before reapplying thevoltage and capturing additional sample material.

A method of nondestructive sampling of intracellular sample materialfrom within a cell at multiple time points may include: applying avoltage between an upper electrode and a lower electrode through atleast one nanostraw in each of a plurality of sample regions of ananostraw substrate to open one or more pores through a cell membraneextending over an opening of each nanostraw; capturing sample materialat each of the plurality of sample regions, wherein the sample materialis released into the nanostraws to a plurality of sample collectorsbeneath the at least one nanostraw corresponding to each of theplurality of sample regions; stopping the application of voltage betweenthe upper and lower electrodes; allowing the cell to recover for aminimum recovery time of at least 1 hour before reapplying the voltageand capturing additional sample material at each of the plurality ofsample regions; and identifying a different biomarker from the capturedsample material for each of the plurality of sample regions at differenttimes.

Also described herein are apparatuses, including systems and devices,for nondestructively sampling intracellular material. For example, asystem may include: a cell culture chamber having an upper region and alower region; a nanostraw substrate positioned over the lower region,wherein the substrate comprises a plurality of sample regions; aplurality of nanostraws extending through the nanostraw substrate ineach sample region, wherein each nanostraw has an outer diameterconfigured to support a cell without penetrating the cell's cellmembrane; a plurality of sample material collectors, wherein each samplematerial collector corresponds to one sample region of the plurality ofsample regions; a first electrode in the upper region; a secondelectrode in the lower region; and a controller coupled to the firstelectrode and the second electrode and configured to apply a pulsedvoltage through the plurality of nanostraws of between about 1 V and100V, a pulse width of between about 10 microseconds and 50 millisecondsfor a duration of between 1 second and 300 seconds.

The nanostraw substrate may comprises a pattern of recessed sampleregions. The pattern may be grid or any other pattern. The recessedsample regions may be recessed on one or both sides.

The nanostraw substrate comprises may be a removable capture substrateconfigured to be removably placed into the cell culture chamber. Thenanostraw substrate may be keyed to fit within the cell culture chamberin a unique orientation (e.g., including a notch, cut-out, protrusion,or the like, and/or having a shape) that requires that the substrate beoriented in a specific configuration so that it can fit into and engagewith the cell culture chamber.

The nanostraw substrate may be formed of any appropriate material; forexample, the substrate may comprise a polycarbonate membrane.

The nanostraw substrate may comprise a blocking coating covering thesurface of the nanostraw substrate between the sample regions, such as ablocking polymer coating. Thus, only the sampling regions may includenanostraws (or “open” nanostraws). In general, the thickness of samplesregions of the nanostraw substrate is between 10 nm and 5 microns (e.g.,less than 5 microns, less than 3 microns, less than 2 microns, less than1 micron, etc.).

The lower region of the cell culture chamber may include a plurality ofsample ports, wherein each sample material collector is associated witha unique sample port. For example, the lower region may correspond tothe sample material collectors; in some variations the sample collectorsmay be separate from the bottom of the cell culture chamber and/orinserted into the cell culture chamber. If the sample materialcollectors are configured to collect material in liquid suspension, thematerial collector may include a fluid containing/storage region inaddition to or instead of a sample port.

The nanostraws may be any appropriate size, typically for making contactwith the cells without penetrating them. For example, the nanostraws mayhave an outer diameter between about 20 nm to about 1500 nm (e.g.,between 100 nm and 1500 nm, between 150 nm and 1500 nm, greater than 150nm, greater than 160 nm, greater than 170 nm, greater than 180 nm,greater than 190 nm, greater than 200 nm, etc.). For example, eachnanostraw may have an outer diameter of greater than 100 nm.

The nanostraw may be made of any appropriate material, particularlynon-sticky materials, such as alumina.

Each of the plurality of sample material collectors may comprises asample material capture substrate configured to bind to the samplematerial (e.g. solid phase substrate, substrate to which a biding agenthas been attached, membrane, including charged membrane, etc.). Thesample material collectors may be removable.

The second electrode may be positioned between the nanostraw substrateand the plurality of sample material collectors. Alternatively, theplurality of sample material collectors may be positioned between thenanostraw substrate and the second electrode.

The plurality of sampling regions may each be configured to have amaximum diameter of between 5 μm and 200 μm (e.g. between 5 μm and 150μm, between 5 μm and 100 μm, etc. e.g., less than 200 μm, less than 150μm, less than 100 μm, less than 75 μm, less than 50 μm, less than 30 μm,less than 20 μm, less than 15 μm, less than 10 μm, etc.).

For example, a system for sampling intracellular material may include: acell culture chamber having an upper region and a lower region; ananostraw substrate positioned over the lower region, wherein thesubstrate comprises a pattern of recessed sample regions; a plurality ofnanostraws extending through the nanostraw substrate in each sampleregion, wherein each nanostraw has an outer diameter configured tosupport a cell without penetrating the cell's cell membrane, wherein theouter diameter is between about 20 nm to about 1500 nm; a plurality ofremovable sample material collectors comprising a sample materialcapture substrate, wherein each sample material collector corresponds toone sample region of the plurality of sample regions; a first electrodein the upper region; a second electrode in the lower region; and acontroller coupled to the first electrode and the second electrode andconfigured to apply a pulsed voltage through the plurality of nanostrawsof between about 1 V and 100V, a pulse width of between about 10microseconds and 50 milliseconds for a duration of between 1 second and300 seconds.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe claims that follow. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIGS. 1a-1f illustrate one example of a nanostraw (NS) extractionsampling apparatus (e.g., system, device, etc.). FIG. 1a shows anexample of a system using a polymer membrane with protruding nanostrawshaving an outer diameter of about 150 nm. This membrane connects thebottom of a chamber (e.g., a 5 mm glass cylinder) to an extractionbuffer. For sampling, the NS well is removed from the incubator andplaced on an electrode (e.g., an ITO electrode), as shown. A smallquantity of the intracellular contents are sampled using an electricalpulse (or pulse train) to open holes in the cell membrane, allowingmaterial to diffuse through the NS into the extraction buffer (pink). Analiquot of the buffer may then be aspirated with a standard pipette andanalyzed conventionally, e.g., using fluorescence imaging, ELISA, qPCR,etc. FIG. 1b illustrates the active sampling using the apparatus shownin FIG. 1 a. During sampling, intracellular species within the celldiffuse through the NS and into the extraction buffer below themembrane. In this example, the sampling region may be defined by arecessed region in the substrate (e.g., the NS membrane, shown here as apolycarbonate membrane). The size of the sampling region can be defined(e.g., lithographically) such that only the cells that grow in theactive regions are sampled. FIGS. 1c and 1d show 45° tilted viewscanning electron microscope (SEM) images of an approximately 150 nmdiameter NSs and a 200×200 μm active sampling region using a systemconfigured as shown in FIGS. 1a -1 b. In this example, it wasdemonstrated that cells outside this window are relatively unaffected bythe sampling process. FIGS. 1e and 1f show exemplary SEM images of cellscultured on (in FIG. 1e ) a 50×50 μm active sampling region containing42 cells, and (in FIG. 1f ) a 15×15 μm sampling region used to isolateand sample from a single cell.

FIGS. 2a-2d illustrate an example of the methods and apparatusesdescribed herein used to provide longitudinal sampling of GFP/RFP fromthe same subpopulation of CHO cells. FIG. 2a shows fluorescentmicroscopy images of GFP (green channel, top) and RFP (red channel,bottom) of a culture of 38 cells on a 200×200 μm NS sampling region(dashed squares). Images were taken every 4 h just before the nanostrawextraction method (NEX) sampling method described herein was performed.RFP transfection was performed after the 8 h time point. FIG. 2b showsnormalized cellular GFP contents from fluorescence microscopy comparedto the NEX extracted GFP quantities. No statistically significantdifference was observed between the relative extracted GFP level andrelative intracellular GFP expression level from cells for each timepoint. Uncertainty bars reflect standard deviation of underlying signal(p>0.05 for both factors, two-way ANOVA). FIG. 2c shows normalized RFPfluorescence intensity compared to the extracted quantities. A smallbackground signal was present before RFP transfection, yet the clearincrease of extracted RFP correlated with the actual increase in RFPexpression. Error bars denote s.d. (p<0.05 between time points, p>0.05for extracted to fluorescence, double asterisk indicates P<0.01, tripleasterisk indicates P<0.001, post-hoc Tukey test). FIG. 2d shows cellviability as a function of time (sampling points). Cultures showed >95%viability immediately after sampling, and >100% over time as the cellsdivided.

FIGS. 3a-3e show longitudinal sampling of RFP from a single CHO cell.FIGS. 3a-3d show fluorescent microscopy images of a single RFPexpressing cell on a 100×100 μm sampling region (dashed squares). RFPtransfection was performed after the day 2 sample point. FIG. 3e showscalibrated RFP quantities in the cell from fluorescence compared to theextracted quantities. The extracted RFP amounts followed the increase inRFP expression within the cell.

FIGS. 4a-4d illustrate the sampling spatial distribution using themethods and apparatuses described herein. In FIGS. 4a and 4b ,fluorescent microscopy images of GFP of a culture of 26 cells on a200×200 μm NS sampling region (white dashed squares) are shown. FIG. 4ashows GFP-expressing CHO cells before sampling, and FIG. 4b showsGFP-expressing CHO cells immediately after sampling. Locally diminishedGFP intensities (dark spots, see, e.g., FIG. 10, below) were observed inthe cells after sampling, likely corresponding to the locations whereGFP was removed from the cells. Scale bar is 50 μm; the brightness hasbeen increased to highlight the spots. FIG. 4c shows a diagram of afinite element model of sampling through the NS. In this example, thecell was treated as a 20×20 μm source that is 1 μm tall, connected tothe extraction buffer by varying numbers of 14 μm long, 150 nm diameterNS. FIG. 4d illustrates an example of the percentage of the cell'sinitial GFP that diffuses into the extraction buffer as a function oftime and number of NS, when modeled similar to FIG. 4 c.

FIGS. 5a-5c illustrate one example of longitudinal sampling from humaninduced-pluripotent stem cells (hiPSC) derived cardiomyocytes andastrocytes. FIGS. 5a and 5b show longitudinal HSP27 extraction from thesame hiPSC-CMs for 4 days. In FIG. 5a , the cardiomyocytes werestimulated by increasing the temperature to 44° C. for 30 min beforesampling at day 2. An up-regulation of HSP27 was observed at day 3 (n=4,the asterisk indicates P <0.05, Tukey posthoc test, one-way ANOVA). TheHSP27 level started to drop at day 4. In FIG. 5b , non-heat shockedhiPSC-CMs were longitudinally sampled for four days (n=4, P >0.05,one-way ANOVA test). FIG. 5c shows representative images of astrocytesderived from hiPSC in 3D cultures (hCS) and cultured in monolayer on NS.Astrocytes are labeled fluorescently with a lentiviral reporter(hGFAP:eGFP) and immunostained with an anti-GFAP antibody. Themorphology of astrocytes was maintained even after 20 days of cultureand repeated sampling on the NS platform.

FIGS. 6a-6e illustrate mRNA expression level in extraction fromhiPSC-CMs using the methods described herein. In FIG. 6a , mRNAexpression level in NS-extraction (black) and from lysed cells (grey) indelta Ct (normalized to GAPDH). A higher delta Ct represents a lowermRNA expression, and the baseline delta Ct was set to 40 cycles toindicate no expression. Of the 48 genes (see, e.g., the table in FIG.16, listing the size and name of the 48 analyzed genes) examined, sevengenes were found to be statistically different from the lysis control(n=4 for living cell extraction, n=2 for cell lysis, single asteriskindicates P<0.05, double asterisk indicates P<0.01, t-test with 95% CI,pooled standard deviation. In FIG. 6b , mRNA sizes of matched andunmatched genes (single asterisk indicates P<0.05, t-test) shows aslight detection difference between larger and smaller genes. FIG. 6cshows a correlation of delta Ct of the 40 matched genes in living cellextraction with the mRNA expression level in cell lysis (R=0.89,p<0.0001, black dots: matched genes, gray dots: unmatched genes). InFIG. 6d , longitudinal mRNA expression level of ˜15 hiPSC-CMs on 100×100μm NS platform over 3 days is shown. Fifteen of eighteen genes showedconsistent gene expression, showing the reliability of the samplingprocess. Error bars are the SD of technical duplicates. In FIG. 6e ,microscopy image of ˜15 hiPSC-CMs after day 3 sampling is shown. Thesecells were actively beating, yet could still be successfully sampled.

FIGS. 7a-7f illustrate one example of a technique for forming patternednanostraws (NSs) as part of a substrate for any of the apparatusesdescribed herein. In this example, a patterned NS sampling platform wasfabricated starting with (in FIG. 7a ) a track-etched polycarbonatemembrane having a 150 nm pore diameter and pore density of approximately1×10⁸ pores/cm² (any appropriate pore diameter, including, e.g., between1×10⁴-1×10⁹ pores/cm², may be used). In FIG. 7b , a 10 nm film ofaluminum oxide was coated on all the nanoporous surface of thepolycarbonate membranes, in this example by atomic layer depositionusing trimethyl aluminum and water as precursors. A pulse form of a-b-cwas used for deposition, where a is the precursor exposure time (0.025s), b is time for the precursor to retain in the ALD chamber (30 s), andc is the N₂ purge time (30 s). In FIG. 7c , a 5 μm thick positivephotoresist film was spin-coated on the top surface of the ALD coatedpolycarbonate membrane, e.g., using a spinning speed of 3500 rpm for 60s. The photoresist-coated membrane was baked at 95° C. for 2 min toevaporate resist solvent. In FIG. 7d , the photoresist-coated membranewas exposed to a square pattern of intense UV light for 5 s, and thenwas developed by immersing into the developer for 60 s. These two stepscreate a sampling region. In FIG. 7e , after photolithography, thealuminum oxide surface in the microwell was etched away, e.g., by aPlasma Quest reactive ion etcher (RIE) with a fast flow composition of40 sccm BCl₃, 30 sccm Cl₂, and 5 sccm Ar at 300 watts at roomtemperature, leaving a polycarbonate surface inside the microwell.During the RIE, the unexposed photoresist served as a protection layer.Because the photoresist is much thicker than the alumina layer, thealumina layer inside the microwell can be fully remove without affectingthe overall structure of microwells. In FIG. 7f , Oxygen RIE with 30sccm O₂ at 300 watts was used to selectively etch the polymer until thedesirable height of NS (e.g., between 10 nm to 10 μm, between 20 nm to 3μm, etc., between 10 nm to 2 μm, between 10 nm to 1.5 μm, etc.) wasobtained. The etching rate of alumina from oxygen RIE is slower comparedto the etching rate of the polymer, thus the etching of alumina isnegligible. The etching rate of both photoresist and polycarbonate arealmost the same, so to make 1 μm tall nanostraws, 1 μm of photoresistwill also be etched way. The photoresist was thick enough to allowfabrication of 3 μm nanostraws inside the microwell without completelyremoving the photoresist during etching. Thus, the photoresist can stillact as blocking layer to cover the pores outside the microwell.

FIGS. 8a-8f illustrate one example of a method (and calibrationtechnique) that may be used in conjunction with the methods andapparatuses described herein. In FIGS. 8a -8 f, isotachophoresis (ITP)was used to concentrate GFP and RFP at 10,000 times. ITP is a form ofelectrophoresis that focuses the target molecules in a focusing zonebetween two electrolytes with different ionic mobility (including g aleading and trailing electrolyte, LE and TE, respectively). After thecomplete formation of the ITP focusing zone, total FP mass can bequantified by measuring fluorescent intensity of the ITP zone imagesobtained with a fluorescence microscope and a CCD camera. In FIGS. 8a ,3 to 10 μL of LE was injected at the LE reservoir and filled the entirechannel. 1 to 5 μL of the sample and TE mixture was injected in the TEreservoir. In FIG. 8b , 1100V DC was applied between the two Ptelectrodes placed at TE and LE reservoirs. In FIG. 8c , 2 min after theformation of ITP zone, the fluorescence intensity of the ITP zone wasstabilized, indicating complete focusing of all GFP molecules in themicrofluidic chamber. In FIG. 8d , time-resolved images showing theformation of an ITP zone (the white dash line indicates channel edges),at increasing distances in the microfluidic channel are indicated in thefigure. In FIG. 8e , GFP intensity in the ITP zone over time (increasingdistance) is shown. The fluorescent intensity value at each distance wasextracted by averaging the intensity of ITP zone , as shown in FIG. 8f ,showing GFP and RFP standard curves generated based on the measuredintensity (n=3) from GFP and RFP standards diluted in the TE buffer.

FIG. 9 shows another example of longitudinal sampling. In FIG. 9,longitudinal sampling of GFP/RFP is illustrated from the samesubpopulation of GFP expressing CHO cells. Fluorescent microscopy imagesof GFP (green channel, top) and RFP (red channel, bottom) of a cultureof 48 cells on a 200×200 μm² NS sampling region (dashed squares) isshown. Images were obtained every 4 hours just before the NS samplingprocess was performed. RFP expression was observed starting at the 12 htime point, 4 hours after RFP transfection using lipofectamine.

FIG. 10 illustrates “dark spots” (regions where an intracellularcomponent being sampled was diffused away by the method describedherein). In FIG. 10, fluorescent microscopy images of GFP of a cultureof 26 cells on a 200×200 μm NS sampling region (dashed squares) isshown. The dark spots as obtained by manual counting were labeled in redto indicate their location in cells.

FIGS. 11a and 11b illustrate a linear relationship between GFP/RFPconcentration and intensity using the methods described herein. GFP andRFP solutions with concentration of 0.25, 0.13, 0.07, 0.04, and 0.02pg/ml, were injected in a 10 μm wide and 12 μm deep glass microfluidicchamber. Microscopic fluorescence images of the GFP/RFP filledmicrofluidic chamber were obtained using (in FIG. 11a ) 200 ms and (inFIG. 11b ) 25 ms exposure time. The fluorescence intensity of theGFP/RFP solutions was measured (n=3, error bars show standarddeviation).

FIGS. 12a and 12b show SEM images of cardiac cells on NS platform. InFIGS. 12a -12 b, hiPSC-CMs are spread out on the NS substrate platform.As observed, the NS are not broken off by the beating of thecardiomyocytes, but instead, the nanostraws bend to accommodate thestress. Note the cells are dehydrated during the SEM preparationprocess, and thus the images are not indicative of cell morphology inculture. Instead, these images show the NS survive after culture withbeating cardiomyocytes. Scale bar in FIG. 12a also applies to FIG. 12 b.

FIGS. 13a-13b illustrate the use of the methods and apparatusesdescribed herein with iPSC-derived astrocytes and neurons generated in3D hCS and cultured in monolayer on NS. In FIG. 13a , astrocytes arelabeled with the hGFAP::eGFP reporter and co-immunostained with ananti-GFAP antibody. In FIG. 13b , neurons are immunostained with ananti-MAP2 antibody, and are shown to not co-localize withhGFAP::eGFP+cells. Scale bars: 50 μm.

FIG. 14 graphically illustrates the co-localization of mRNA using themethods as described herein. In FIG. 14, intracellular localization ofmatched and unmatched mRNA is shown. Subcellular localization of themRNA may affect the sampling efficiency. To examine this, mRNA wasassumed to be localized to the same regions as the proteins that theycode for. The protein localization was then compared in 12 subcellularlocations of the specific unmatched gene to the matched gene. mRNAs thatcode for proteins found in the plasma membrane were statistically shownto be more often unmatched (P<0.05, t-test), which indicate that thespecific mRNA sequences is more likely immobilized within the cytoplasmleading to low detection rate.

FIG. 15 graphically illustrates a comparison between detected levels for48 different genes as sampled by the methods described herein (e.g.,nanostraw extraction/diffusion “living cells,” control lysed cells andnegative control (without electrical pulsing)). The heat map shown inFIG. 15 illustrates that the cytoplasmic level of 48 genes from livingcells matched well with control (cell lysis) results. Very low or nogene signal was detected in the negative control collected after washing(without electroporation using the nanostraw extraction), indicating thewashing step is sufficient to remove the background contamination.

FIG. 16 is a table illustrating the sizes and names of the 48 analyzedgenes (highlighted genes indicates unmatched genes).

FIG. 17a shows a first example of an apparatus configured for spatialsampling (and/or sampling over time) of intracellular material using thenanostraw sample extraction methods described herein. In FIG. 17a , acapture substrate/surface is placed below the nanostraw substrate (andmay be swapped out/replaced at different times) to capture or collectthe material being sampled from within the cell. In FIG. 17a , thebottom electrode is placed behind the capturing substrate/surface.

FIG. 17b shows another example of an apparatus configured for spatialsampling (and/or sampling over time) of intracellular material. In thisexample, the capture substrate/surface may be at the bottom of theapparatus (and removal swapped out) and the bottom electrode may beabove the capture surface (e.g., adjacent to the base of thenanostraw(s).

DETAILED DESCRIPTION

In general, described herein are methods and apparatuses fornondestructively sampling intracellular material. These methods andapparatuses may be based upon diffusively sampling material from insidethe cell using a nanostraw (NS) embedded substrate. Typically, forexample, cells of interest are cultured on a substrate (e.g., a polymermembrane) containing nanostraws, which may be localized to discreteregions (e.g., defined regions). The nanostraws are hollow and extendthrough the substrate and protrude from the surface of the substrate(see, e.g., FIG. 1a ). For example, in FIG. 1 a, the nanostraw substrate101 is positioned a cell culture chamber 103 having an upper region(cell culture reservoir 105) and a lower region 107 holding anextraction buffer. The nanostraw substrate 101 is positioned over thelower region, and includes one or more active sample regions (see, e.g.,FIG. 1b ). The active sample regions 109 may be recessed. The substratemay have a pattern of such recessed sample regions. The pattern may be agrid pattern or any other arrangement of sample regions.

As shown in FIGS. 1a and 1 b, a plurality of nanostraws 111 may extendthrough the nanostraw substrate 101 in each sample region 115. Eachnanostraw may have an outer diameter configured to support a cellwithout penetrating the cell's cell membrane (e.g., the outer diametermay be between about 20 nm to about 1500 nm, or in particular, greaterthan 140 nm, e.g., 150 or greater, 200 or greater, etc.).

In general, an apparatus such as the system for nondestructivelysampling intracellular material shown in FIG. 1a-1f may include aplurality of sample material collectors for collecting sample materialreleased by the cell when the cell membrane is opened by applying apulsed voltage through the nanostraw. The sample material collector 113may collect liquid (e.g., extraction buffer into which the samplematerial is suspended) and/or it may include a bound sample material.For example, the sample collector 113 may include a sample materialcapture substrate. Any of these systems may include an array of samplematerial collectors, wherein each sample material collector correspondsto one sample region, and the paired sample regions and sample collectormay be isolated from other sample regions and sample collectors.

In general, any of these systems for nondestructively samplingintracellular material may also include a first electrode in the upperregion 117, and a second electrode in the lower region 119. A controller121 may be coupled to the first electrode and the second electrode andconfigured to apply a pulsed voltage through the plurality ofnanostraws. As mentioned, the pulsed voltage may be, for example,between about 1 V and 100V, having a pulse width of between about 10microseconds and 50 milliseconds and may be applied for a duration ofbetween 1 second and 300 seconds. The controller may be specificallyadapted to apply the driving voltage within this range of values, andmay be adjustable. For example, the user may adjust the applied peakvoltage, pulse duration, and/or duration that the pulsed voltage isapplied. The controller may also limit (e.g., prevent) the apparatusfrom applying additional voltage until after some minimum recovery time,which may be pre-set (e.g., to 4 hours or more) or may be user-selectedto a value that is, e.g., between 1 hour and 48 hours, during whichtime, further applied voltage may not be applied.

When the cells are cultured on the substrate, the cells may grownormally over the entire substrate (e.g., polymer membrane), such thatcells within the sampling region interact with surrounding cells,avoiding cell isolation. Intracellular samples may be collected byapplying an electrical voltage through the nanostraws (NSs), locallyopening small holes in the cell membrane near the NS tip. The appliedenergy may be configured such that, during the subsequent interval(e.g., typically between 2 to 5 min) when these pores are open, ˜5-10%(e.g., less than 15%) of the proteins, mRNA and small molecules maydiffuse and/or migrate based on charge from out of the cells, throughthe NS, and into an extraction solution below the culture well (see,e.g., FIG. 1b ). Any appropriate extraction buffer may be used as longas it has a reasonably osmolality-matched to the cell; typically, e.g.,1× phosphate-buffered saline solution (PBS) may be used. After thisinterval (e.g., the 2-5 min interval), the sample material released fromwithin the cell and into the nanostraw that is collected in a samplecollector beneath the nanostraw (e.g., in the extraction buffer and/orany solid phase support) may be removed from the sample collector andanalyzed conventionally, including fluorescence, mRNA detection, orELISA assays. The cell culture well may then returned to an incubatoruntil a new sample is required. In some variations the samplecollector(s) under the nanostraws may be removed and/or replaced.

The methods described herein (which may be referred to herein as the NEXprocess) may be used to extract, evaluate and analyze one or morepreferably many different intracellular components (e.g., protein and/ormRNA contents) both statically and longitudinally. These methods andapparatuses have been found to be nondestructive and may providequantitatively useful information about intracellular contents for mRNAsequences and proteins. Notably, the methods and apparatuses describedherein had >95% cell viability that enabled multiple, real-time samplingover extended time periods, and was well tolerated over 20 days by humanastrocytes derived from hiPSCs. Equally important is the samplingprocess extracted species throughout the cell, providing a comprehensiveview of expression rather than a single site extraction location. Thesystem may be used for some, but not all, larger nucleic acid molecules(>15,000 nt) even despite slower diffusion and limited cytosolicaccessibility. NEX sampling was successful even for single cells,although the small quantities of material extracted at this levelrestricted applicable analytical methods. Overall, the NEX processappears to be a straightforward method to non-destructively followtemporal dynamics of cellular protein and mRNA contents over time.

The NEX platform described herein may be based on a substrate includinga (e.g., polycarbonate membrane) including a plurality of nanostraws. InFIG. 1 c, for example, the NSs have an approximately 150 nm outerdiameter, forming an inorganic NS extending through the polymer andprotruding 1-3 microns above the surface. In FIG. 1a and 1 b, this NSmembrane is mounted on the bottom of a 2-5 mm diameter glass cylinder101 that fits into a 48- or 96-well plate for cell culture. Fabricationof the NS membranes is illustrated and described in FIG. 7a -7 f, inthis example, producing flat polycarbonate membranes with NS extendingfrom the surface (see, e.g., FIG. 1c ), where height is readilycontrollable. Specific cell-sampling regions may be defined, e.g., byblocking the remainder of NS membrane with blocking coating such as aphotolithography-patterned polymer (See, e.g., FIGS. 7a-7f ). Duringcell culture, only the cells that grow in the selected regions withexposed NS will be sampled, leaving cells on the blocked area unaffected(FIGS. 1d and 1e ). The size of the sampling window can be adjusted from<1 micron on a side to millimeters, allowing scalable sampling from asingle cell to a hundred thousand, while maintaining cell-to-cellconnectivity and communication (e.g., FIGS. 1e and 1f ).

Cells grown on the NS described herein have been found to demonstratenormal cell behavior and mRNA expression , as shown in FIG. 1 c.Typically, for many cell types, 100 nm or smaller diameter NSspontaneously penetrate the cell membrane, allowing delivery of smallmolecules into cells. Larger NS (e.g., 110 nm or larger, 120 nm orlarger, 130 nm or larger, 140 nm or larger, e.g., such as 150 nm andlarger) are instead engulfed by the cell membrane without causingmembrane rupture. However, access to the cytoplasm can still be gainedby applying short electric pulses (e.g., pulses of between about 10-35V) to temporarily open small pores on the cell membrane at the NS-cellinterface. The energy applied may be configured so that two to fiveminutes after the pulses, the cell membrane recovers and the cellsevolve unperturbed. In order to prevent systemic cytosolic leakage, insome examples (e.g., using between about 110 and 1000 nm diameter, e.g.,about 150 nm diameter NS) the use the electrical pulsing maybecontrolled a ‘valve’ to gate sampling. The methods and apparatuses maytherefore titrate when cells release contents through the NS, whilemaintaining their membrane integrity throughout the remaining cultureperiod.

These methods and apparatuses may therefore allow real-time,longitudinal sampling from cell subpopulations and single cells, asdescribed in FIGS. 2a -2 d. In this example, the NEX sampling processwas evaluated for quantitative analysis of intracellular proteinconcentrations within the same set of cells over time. A level of GFPfluorescence in NS-derived samples was extracted and measured andcompared these values with the GFP fluorescence of the sampled cells.GFP-expressing CHO cells were cultured on the NS membrane with a 200×200μm active area that mounted on a 2 mm glass cylinder. Cells were sampledevery 4 h for 16 h total (e.g., at five time points, see FIG. 2a ,columns). Dynamic changes in expression were examined bylipofectamine-transfecting with a plasmid containing RFP at the 8 h timepoint, for which expression became observable at 12 and 16 h. At eachsampling point, the NS well was removed from the incubator, washed withPBS to remove possible contaminants, and the GFP and RFP intensity ofthe cells on the NS window was measured with fluorescent microscopy(FIG. 2a ). In this example, a series of short electrical pulses wereapplied for 20 s, opening small holes in the cell membrane at the NStips, and the cellar proteins were allowed to diffuse through the NS andinto the extraction buffer for 10 min The NS well was then returned tothe incubator, and the amount of GFP/RFP in the extraction buffer wasanalyzed with fluorescence using isotachophoresis (ITP) to selectivelyconcentrate the proteins (see, e.g., FIG. 8a-8f for a description ofthis technique). Normal cell morphology was observed throughout theexperiment, and cell viability was >95% per sampling on average (FIG. 2d) indicating the cells were healthy during and after the samplingprocess. Experiments on sister cultures (e.g., FIG. 9) did not showqualitative differences.

FIG. 2b shows the quantitative comparison of the cells' GFP fluorescenceby microscopy, and the NS-extracted GFP/RFP intensities from the 38cells in the active NS region. The measurements were normalized to thehighest value in each run in order to account for the different numberof cells present, and averaged to provide standard deviations. The meanGFP expression level in the sampled cells did not show a significantchange, as expected for a stably expressing protein. The NEX extractedGFP accurately followed this trend. The relative NEX-measured GFP levelsdid not show significant statistical difference (Two-Way ANOVA) with theGFP expression level in cells (p >0.05 for both time and extracted tofluorescence comparison) at any of the five time points. The extractedGFP signal was however significantly lower at the first time point,which was systematically observed for all NEX experiments, suggestingthe initial extraction is less efficient. Thus, while not rising to thelevel of statistical deviation, the initial data point should usually bediscarded, though we show all samples in this work. See, e.g., FIG. 2a-2 d.

The NEX methods described herein (e.g., methods for nondestructivesampling of intracellular sample material from within a cell) can alsofollow temporal dynamics, namely the change in RFP as the cells begin toexpress RFP fluorescent proteins after transfection (see, e.g., FIG. 2c). Extracted RFP levels were equal to the background fluorescence forthe first 3 time points, then increased quickly at 3 and 4 days, inagreement with microscopy images (p<0.001, Two-way ANOVA). Nosignificant difference between NEX extracted amounts to the fluorescenceimaging was observed (p>0.05, Two-way ANOVA). The sampling process couldthus also measure dynamic changes in cell expression over time.

Encouraged by the results on this subpopulation of 38 cells, the activeNS area was reduced to 100×100 μm to sample a single cell (See, e.g.,FIG. 3a-3e ). In this example, the cell was sampled once a day for a4-day period and RFP contents were analyzed using ITP (see, FIG. 3a-3d )and compared to fluorescence microscopy images. After sampling at day 2,the cells were transfected with an RFP plasmid using lipofectamine. Onecell in the NS active area fluoresced on day 3, and intensified on day4. The absolute quantity of RFP may be determined, e.g., using acalibration curve (See FIGS. 11A-11B) for both the microscopy and NEXmeasurements, allowing direct quantitative comparison. FIG. 3a-3e showsthe calibrated mass of cellular and extracted RFP from a single cell.The extracted RFP expression trend and the actual cell concentrationwere in good quantitative agreement relative to their initial baselines.The total RFP mass inside the cell was 1.7 pg and 2.0 pg at day 3 and 4,respectively, compared to 120 fg and 150 fg for the extracted RFP atthose sampling points. This corresponds to an extraction yield of 7% and8% of the total cellular RFP at the third and fourth sampling points,respectively.

The NEX process is configured so that only extracts a fraction of thetotal contents of the cell (e.g., 15% or less), hence the reason it isnon-destructive, and therefore a relative calibration is necessary toinfer absolute intracellular concentration. However, the extractionquantities over repeated sampling in FIGS. 2a-2d and 3a-3e show thateach sampling event is highly consistent, and although extractionpercentages vary somewhat from cell to cell the longitudinal extractionquantities are precise. For example, in two different single cellmeasurements one cell may give a 5% extraction efficiency and another10%, yet each sampling event consistently yields the same percentagefrom each cell (e.g., FIG. 3e ). Thus the method is capable of not onlydetecting the presence of an analyte, but reliably quantifying thecytosolic quantities over time.

The apparatuses and methods described herein may also allow for spatialdistribution and efficiency of sampling. The NEX process may beconfigured to reflect the contents of the entire cell, or samples only asingle site. The spatial distribution of NS extraction from the decreaseof GFP intensity within GFP-expressing CHO cells during sampling wasassessed. CHO cells were cultured overnight on a patterned membrane with200×200 μm region of ˜40,000 exposed NS (see, e.g., FIG. 4a ). Duringthe 2 min sampling period, GFP diffuses out from the cells and throughthe NS, leaving a lower fluorescence intensity region (dark spots) inthe cell where the membrane was opened (FIG. 4b ). The location andnumber of ‘open’ NS can therefore be visualized by the dark spots incells. Twenty-four out of twenty-six CHO cells showed spots duringsampling demonstrating that most cells within the sampling region arepenetrated and sampled through the NS. The multiple penetrating NS (darkspots) were observed to be distributed throughout the cell bodies, withlittle difference between the soma and peripheral regions. NEX thusappears to sample from all regions of the cytoplasm, providing acomprehensive view of the intracellular contents. See, e.g., FIGS. 4a -4d.

The total GFP extracted from the cells during sampling could be measuredfrom the fluorescence intensity difference before and after sampling.The average GFP in a cell before and after sampling in this example wasapproximately 0.50 (±0.44) pg and 0.47 (±0.38) pg, calculated from acalibrated volumetric GFP intensity curve (See FIGS. 11a-11b ). The GFPextracted from these 24 cells was 680 fg from the change in fluorescenceintensity, or 6% of the initial cell concentration. This fraction isalso similar to the single cell extraction percentages in FIGS. 3a-3d(7% and 8%). Since we know the amount of material the cells lost, wealso calculated the collection efficiency. The calibrated amount of GFPmeasured in the extraction buffer during this same experiment was 230fg, or ˜30% of the total amount lost from the cell. This is reasonablecollection efficiency, indicating the material loss during extractionand handling is not limiting. Together, these results show that most ofthe cells within the sampling region were extracted from, that multipleNS penetrate the cell at one time, that molecules were sampled frommultiple regions of a cell, and that cell contents are extracted andanalyzed with a reasonable collection efficiency.

In theory, the amount of material extracted may be a function of thecellular concentration, the diffusivity of the species, and the NSgeometry . The extraction was simulated as a purely diffusive transportprocess using a finite-element model (COMSOL Multiphysics, Palo Alto,Calif.) of a cellular volume (20 μm diameter; 1 μm tall), connected tothe 1× PBS extraction buffer through a set of 14 μm long, 150 nmdiameter NS (FIG. 4c ). The expected percentage of the total GFPextracted from the cell as a function of time for a GFP diffusivity of87 μm/s is shown in FIG. 4d , and agrees with our experimentalobservations. For 6 penetrating NS, close to the observed number ofspots per cell (FIG. 4b ), ˜9% of the total GFP diffuses into theextraction buffer over the 2 min extraction interval, which correspondswell with the 7% and 8% GFP measured from the single cell experiments.See, e.g., FIGS. 5a -5 c.

The apparatuses and systems described herein may also permitlongitudinal sampling of proteins from hiPSC-derived cardiomyocytes andastrocytes. For example this apparatus, including the apparatus andmethods described in FIGS. 1a-4d illustrate the operation of the methodsdescribed herein.

The NEX methods can be used to sample contents not just from cell linesbut also for cell types derived in vitro from human induced pluripotentstem cells (hiPSC), which is essential in future applications related tocell differentiation and disease modeling. We assessed longitudinalextraction and off-platform analyses of non-fluorescent heat shockprotein 27 (HSP27) from hiPSC-derived cardiomyocytes (hiPSC-CMs),measured with ELISA (FIG. 5a-5c ). Heat shock protein is upregulatedwhen exposed to external stressors, and is thus suitable for studyingtransient processes, as descried herein.

In one example, we increased the hiPSC-CM plating on our NS platform to100,000 (±25,000) cells due to the detection limit of HSP27 ELISA (10.9pg/ml, Affymetrix, San Diego, Calif.), which is not sensitive enough todetect the intracellular extraction from small cell populations. See,e.g., FIGS. 12A-12B. After 7 days in culture on the NS, the hiPSC-CMsbegan beating. Even under stress from the continuous beating, the NS didnot break nor were pulled from the cells, allowing sampling even fromthis actively moving tissue (FIGS. 13A-13B). Intracellular extractionswere obtained every 24 h for 5 days. At day 2, the cells were stressedby exposure to a heat shock (44 C for 30 min), which is expected toupregulate the synthesis of HSP27. A sister culture not exposed to theheat shock perturbation were sampled at the same time points as anegative control.

The NS platform followed the temporal expression and upregulation of HSP27 in human CMs. Starting with a relatively low concentration at day 1,there was a small but not statistically significant HSP27 level increase2 h after heat shock perturbation at day 2, suggesting delayedexpression of HSP27. At day 3, the HSP27 increased about 5 times higherthan at day 1 and 2 (n=4, P <0.05, one-way ANOVA), and then decreased atday 4 and 5. In contrast, the HSP27 level of the control was relativelyconstant all four days (n=4, P >0.05, one-way ANOVA test). The firstextraction point showed lower extraction levels in both sets of data,similar to what was observed for the GFP sampling experiment. The smallupregulation of the HSP27 in the control samples indicates minimalstress response due to sampling, and Calcein AM labeling confirmed >90%cell viability for both sample and negative control. These resultsdemonstrate the feasibility to use the NS to extract and measurenon-fluorescent proteins from beating hiPSC-CMs.

In order to assess the influence of the long-term culture on the NSplatform and repeated sampling process on neural cell types, we examinedthe viability of astrocytes derived from hiPSC in 3D cortical spheroids(hCSs) . Approximately 50,000 astrocytes and neurons derived in 132-dayold hCS were plated on the NS platform (FIG. 6c ), and electricallyporated using the same protocol for the cardiomyocytes once per day for20 days. Astrocytes were fluorescently labeled with a cell-specificreporter (hGFAP::eGFP) as previously described. Cell morphology wasfollowed every day during the sampling period, and despite their highoverall reactivity to various stimuli and cell injuries, humanastrocytes cultured on the NS tolerated the platform and the dailysampling well, with insignificant morphological changes between day 1and day 20.

The methods and apparatuses described herein were also used to detectand/or measure mRNA expression levels in human iPSC derivedcardiomyocytes, for example the particular mRNA transcriptomics.Measuring the apparent these mRNA expression levels in human iPSCderived cardiomyocytes may prove to be a powerful method to detect geneexpression, cell phenotype, and cell to cell heterogeneity. With theadvent of efficient reverse transcription and single-cell sequencing,multiple mRNA sequences can be simultaneously detected and with highersensitivity than proteins. To test whether the mRNA extracted fromprimary hiPSC-CMs are statistically related to the actual concentrationsinside the cell, we first performed a NEX extraction of mRNA for 2 minand compared it to the mRNA expression for a lysed sister cellpreparation. The NEX extract was pipetted from below the well andamplified with RT-PCR using a random primer and sequenced with asingle-cell BioMark system. The results were compared to the positivecontrol (n=2) obtained by lysing a sister culture of hiPSC-CMs, and anegative control without the electroporation step (n=4). Both of thesecontrols were amplified and analyzed in the same manner as the mRNAsamples. Among the 48 genes, 25 were cardiac-related genes, including aninward-rectifier potassium ion channel (KCNJ2) and several integralmembrane proteins (e.g., PLN, SCN5A), 13 were stem cell differentiationrelated genes and 10 were housekeeping genes.

The mRNA from the NEX extracts were in good quantitative agreement withthe lysis control samples. All genes with non-zero quantities from lysiswere also detected in the NEX extraction, equal to 44 gene detectionsfor each sample. No false positives were observed, as the 4 mRNA thatwere not detected in the positive controls were also not detected in thecell extraction. The delta Ct of each gene (excluding the statisticallyunmatched genes) in the extraction was strongly correlated with thepositive control (FIG. 6c , R=0.89, P<0.0001). Notably even large genessuch as SMAD2 (10,428 nt) were successfully sampled (see the table inFIG. 16). After sampling, the hiPSC-CMs showed healthy morphology andthe green fluorescence from the calcein AM stain indicated >95% cellviability. Negative controls with electric field, but no NS, showed nodetectable signal, as shown in FIG. 14.

Statistical t-test analyses of the 44 detected mRNA sequences found thatonly seven genes were significantly different than the control (P<0.05,t-test) shown as light gray dots in FIG. 4c . The lower detectionefficiency of the seven unsuccessful genes could be due to severalfactors, including lower diffusion rates due to size or binding tostructures within the cell. We did not include a statistical multipletesting correction intentionally because such a procedure, whilemaintaining the overall type I error rate, increases the chance of typeII errors (the chance that differentially extracted genes are notdiscovered). FIG. 6b shows a slight statistical difference between thesizes of the unmatched and matched mRNA (P<0.01, t-test), suggestinglarger size molecules are more difficult to extract during the 2-5 minextraction period, though the result was heavily influenced by RYR2 at16,562 nt. Subcellular localization of the mRNA may also affect thesampling efficiency, as mRNAs that code for proteins found in the plasmamembrane were statistically more often unmatched (FIG. 14, P<0.05,t-test).

The methods (e.g., NEX sampling) described herein may be repeated on thesame set of actively behaving primary cells to provide longitudinal mRNAmeasurements. FIGS. 6d-6e show mRNA expression levels of 18 differentgenes from ˜15 hiPSC-CMs measured once per day for three days. Notethese cells were active and beating at the time they were sampled. Thepresence of additional cell monolayers makes exact cell countingdifficult, however on average 15-20 cells were observed on the 100×100μm sampling region. The measured expression levels were remarkablyconsistent, demonstrating the precision of the sampling and analysisprocess. Of the 18 genes, 15 were highly consistent over three days,implying the variations in MYL7, TNNC1, and ACTB are likely significantand may reflect fluctuations in gene expression. More work is necessaryto definitively establish the biological origin of these fluctuations,yet it is clear the NEX process has the capacity to measure the changeor lack of change in mRNA expression over time from the same set ofcells.

Currently the sensitivity of mRNA sequencing systems are not able tomeasure NEX extracts from single cells, instead requiring ˜15 to 20cells. This agrees well with the ˜7% extraction efficiency,corresponding to ˜1.1 to 1.4 cellular equivalents per sample. With theincreasing sensitivity of single cell mRNA assays this limitation maysoon be overcome, enabling repeated mRNA measurements from a single cellover an extended time period.

Any of the NS substrates descried herein may be patterned andfabricated. The NS membrane may be based on 15 μm (±15%) thicktrack-etched polycarbonate membranes (GVS, Sanford) with 1×10 pores/cm,often used for water filtration and cell culture. A 10 nm thick Al₂O₃layer is deposited on the membrane using atomic layer deposition (ALD)at 110 C, including the insides of the track-etched pores which willbecome the NS walls. The NS are formed by reactive ion etching the Al₂O₃with BCl₃ and Cl₂ in Argon (300 W, 40 sccm BCl₃, 30 sccm Cl₂, 5 mTorr, 5min) from the top surface to reveal the polymer, followed by oxygenplasma etching to remove the polymer and expose the inorganic NS. Tofabricate the photolithographically defined sampling regions, a 5 μmthick positive photoresist film (e.g., MEGAPOSIT SPR2203 i-Linephotoresist, Dow, Austin) was spin coated on the surface of the ALDcoated polycarbonate membrane using a spinning speed of 3500 rpm for 60s. Next, the photoresist-coated membrane was baked at 95 C for 2 min toevaporate resist solvent, and then the photoresist-coated membrane wasexposed to a square pattern of intense UV light for 5 s. After exposure,the membrane was developed by immersing into the MF-26A developer(Shipley, Marlborough) for 60 s. The aluminum oxide surface in thesampling region was etched away by RIE, leaving a polycarbonate surfaceinside the sampling region. Finally, the polymer was etched away byoxygen RIE in order to form the NS.

In any of the methods and apparatuses described herein, prior toextracting material from the cells through the nanostraws as described,the cells may be cleaned to remove excreted material and dead cellfragments. For example the cells may be rinsed in a buffer (e.g.,phosphate buffered saline, PBS). In general, cells may be kept in one ormore different buffers (e.g., PBS or TE buffer), rather than using cellmedia, and rinsed to remove media prior to sampling. Surprisingly, theuse of cell media resulted in an apparent contamination of the sampledportion (e.g., the captured sample material). This may be due toproteins and sugars contained in cell media which could show up ascontaminants in the sampled contents. After sampling, cells may again bebathed in media. Thus, any of the apparatuses described herein may beconfigured to rinse and/or replace the solution (e.g., switching betweencell media and media-free buffers) between sampling. Thus the cellculture chamber of the apparatus may include ports for adding/removingthe material surrounding the cells growing on the substrate; theapparatus may include tubing and/or pumps to add and/or remove (andswitch between the media and buffer) automatically prior to and/or aftersampling.

In any of the methods and apparatuses described herein, as alreadymentioned, the substrate containing the nanostraws may include aplurality of different sample regions. For sampling, these sampleregions may be defined (e.g., lithographically defined) sample region,and may be marked, or visible. Sample regions may be recessed relativeto the rest of the substrate onto which the cells are grown. Opennanostraws for sampling may be present only in the sample regions.

Also, in any of these methods and apparatuses, the polarity of theelectric field may be reversed (e.g., compared to delivery of materialinto the cells) such that negatively charged species will be mobilizedout from the cells.

Any of the apparatuses and methods of using them descried herein mayinclude a sample preparation for scanning electron microscopy: forexample, a NS membrane may be prepared for SEM imaging by sputtercoating with about 10 nm of Au/Pd. Biological samples were prepared byfixing in 2%Glutaraldehyde with 4%Paraformaldehyde (PFA) in 0.1M NaCacodylate Buffer (pH 7.3) for at least 4 h, next, stain with 1%OsO4 for10 min, and followed by dehydration in a series of 30, 50, 70, 90, and100% ethanol with 10 min of incubation at room temperature for eachsolution. The dehydrated sample was dried by critical point drying in100% EtOH with liquid CO₂, and then sputter coating with about 10 nm ofAu/Pd for SEM. Samples were imaged in a FEI Sirion SEM.

To perform the sampling process described, the cells of interest werefirst cultured on the NS membrane within a 2-5 mm glass cylinder withappropriate cell media on top (FIG. 1b ). This culture tube remainedwithin a 96 (or 48)-well plate in an incubator until a sample wasdesired. The sampling process was performed in five steps: First, thecells were washed with 1× phosphate-buffered saline (PBS) three timesand the cell culture media was changed to PBS to eliminate possiblecontaminants. See, e.g., FIG. 15. Second, the NS cylinder was placed ontop of a droplet of 1-15 μl extraction buffer consisting of PBS, or TEbuffer for the ITP assay (explained in later section) on an indium tinoxide (ITO) electrode (FIG. 1a ). A platinum (Pt) wire was immersed intothe cell culture buffer which will act as the counter electrode. Third,10-45 V (between anode and cathode) square electric pulses were appliedfor 20-60 s (200 μs pulse duration, 20 Hz repetition rate) between thetwo electrodes (across the NS membrane). The pulses temporarily openedpores in the cell membrane, allowing freely diffusing intracellularproteins and mRNA to diffuse through the NS to the extraction buffer onthe underside of the NS membrane. We have observed an increased flux ofmolecules when the polarity of the electric field is the opposite of thecharge of the analytes. When extracting negatively charged molecules(e.g. mRNA), the ITO electrode was kept at positive potential, whereasit was kept negative when extracting positively charged molecules.Fourth, after the electroporation, the sampling device was kept on theextraction buffer for another 0.5-5 min to allow diffusion of additionalcytosolic content. Because the extraction process depended mainly onmolecular diffusion, the total amount of extracted molecules wasexpected to depend on both diffusivity and concentration. Therefore, forextraction of larger molecules, longer diffusion time was expected dueto their lower mobility. Finally, the extracted molecules were collectedbeneath the NS membrane for further analysis by pipetting up theextraction buffer.

The second electrode may be an ITP electrode. ITP was conducted in a 50μm wide, 20 μm deep cross-channel design glass microfluidic chip. Theleading electrolyte (LE) and trailing electrolyte (TE) buffers were 200mM of tris and 100 mM of HCl, and 25 mM of tris with 150 mM of glycine,respectively. 1% Polyvinylpyrrolidone (PVP) was added to both LE and TEto suppress electro-osmotic flow (EOF). All reagents were obtained fromSigma Aldrich, Mo., USA. The TE buffer was also used as the extractionbuffer in cell sampling. To pre-concentrate GFP, first, the microfluidicchannel and LE reservoir were filled with 3 to 10 μL of LE, and then 1to 5 μL mixture of TE and GFP sample solution was injected in the TEreservoir. Next, anode and cathode were placed in the LE and TEreservoir respectively. An electric field with 1100 V (Keithley,Beaverton) was applied between the electrodes. The GFP ITP focusing zoneformed and electromigrated towards anode right after applying theelectric field. The GFP intensity was stabilized 2 min after the ITPstarts. See, e.g., FIGS. 8a -8 f.

To obtain a detectable protein signal, in some variations we increasedthe cell population on our NS platform to 50,000 (±25,000) cells due tothe detection limit of HSP 27 ELISA (10.9 pg/ml, Affymetrix, SantaClara), which is not sensitive enough to detect the intracellularextraction from small cell populations. Intracellular extractions wereobtained every 24 hours for 5 days. Cells were washed in PBS before eachsampling in order to remove loosely adsorbed proteins. At day 2, thecells were stressed by exposing them to a 30 min heat shock at 44 C,which is expected to upregulate the synthesis of HSP 27. The day 2extraction was collected 2 h after the heat shock. Cells not exposed tothe heat shock perturbation were also sampled as a negative control.

RNA Extraction from hiPSC-CMs may be performed using the methods andapparatuses described herein. For example, mRNAs may be extracted usingthe NS followed by amplification and analysis using RT-PCR and qPCR. Inorder to average out stochastic fluctuations associated with smallnumbers of cells, we chose to culture 100,000 (±50,000) cells in a NSwell. The cells were rinsed with PBS buffer to remove extraneous orexcreted material, then sampling was performed for 2.0 min as describedpreviously. Since mRNA rapidly degrades in the presence of RNase,carrier RNA (Sigma Aldrich, St.Louis) and RNase inhibitor (Thermo FisherScientific,Waltham) was added to the extraction buffer to make a mixturewith 1 μg/ml and 1 U/μL respectively before sampling. The mRNAs in theextraction buffer were reverse transcribed to cDNAs with Oligo(dT)20(Thermo Fisher Scientific, Waltham). Next, the cDNAs were pre-amplifiedfor 15 cycles and purified using DNA Clean & Concentrator™-5 (ZymoResearch). The pre-amplified cDNAs were then amplified with the 48 geneprimers and analyzed by qPCR in an integrated fluidic circuits Fluidigmchip following standard protocols (Fluidigm, Palo Alto).

To pre-amplify the extracted mRNA, 0.5 μl DEPC-treated water, 0.5 μlOligo(dT) and 0.5 μl dNTP reagent was mixed with 5 μl mRNA extraction.The mRNA solution was mixed and briefly centrifuged, and then heated at65° C. for 5 min and incubated on ice for at least 1 min. 2 μl 5× SSIVbuffer, 0.5 μl DTT, 0.5 μl RNase inhibitor and 0.5 μl SuperScript IVRTase were added into the annealed RNA solution. The combined reactionmixture was incubated at 50° C. for 20 min, then the reaction wasinactivated by incubating the mixture at 80° C. for 10 minutes. 10 μlPooled assay mix, 20μl TaqMan PreAmp Master Mix was added to thecombined reaction mixture. Sixth, the mixture was preamplified at thefollowing conditions: HOLD for approximately 15˜20 CYCLEs: Temp of about95° C. (10 min), cycled for 15-20 cycles at 95° C. (15 secs) for 60° C.(4 min). Finally, the preamplified cDNA was purified using DNA Clean &Concentrator™-5, eluted in 6 μl DNA Elution Buffer. The preamplifed cDNAwas detected by using Fluidigm Dynamic Array IFC for Gene Expression.

The methods and apparatuses described herein may also be configured toculture and perform GFP extraction and Immunocytochemistry ofhCS-derived astrocytes. Human cortical spheroids (hCS) were derived fromiPSC as previously described. The generation of neurons of deep- andsuperficial-cortical layers is followed by astrogenesis in hCS, andafter ˜10 weeks in vitro cortical neurons are accompanied by a networkof non-reactive astrocytes. For sampling experiments, hCS at day 132 invitro were enzymatically dissociated and plated on NS at a density of500,000-750,000 cells per device. The day after plating, platedastrocytes were labeled with a viral reporter (hGFAP::eGFP). For GFPsampling, cells were maintained on the NS for up to 20 days with dailymedia changes. Short electrical pluses (45 V, 200 us pulse width, and 20s duration) were applied to cells every day. For immunocytochemistry,the cells on NS were fixed with 4% PFA for 10 minutes and immunostainedwith an anti-GFAP antibody to label astrocytes, an anti-MAP2 antibody tolabel neurons (MAP2) and anti-Actin antibody to label filaments.

Measuring dynamic intracellular processes and capturing cellularheterogeneity, especially at the single cell level, has become an areaof active investigation in molecular and cellular biology. Despite rapidtechnological advancement in the sampling modalities and sensitivity,these methods are still limited by the need to lyse the cell for sampleextraction. The methods and apparatuses described herein provide asampling platform based on NS for longitudinal, non-destructiveextraction and quantification of proteins and mRNAs from living cells.The procedure itself is quite straight-forward, locally porating a smallarea of the cellular membrane near the NS and allowing cellular contentsto diffuse into an underlying extraction buffer. The process requiressimple equipment and a common voltage supply, and should be feasible inmost laboratories.

Extraction through passive diffusion as described herein has not beenfound to significantly bias the results to only a few of the possiblespecies. Proteins and mRNA were sampled consistently over a wide rangeof sizes, and a similar percentages of the cellular content as smallerspecies has been found. While there was a slight preference for smallermRNA (see, e.g., FIG. 6b ), this was largely due to the influence ofRYR2; without that data point there would have been no statisticaldifference. The fact that a large fraction (41/48) of the mRNA sequenceswere successfully analyzed suggests that a sufficient number of genescan be monitored to make meaningful biological assessments.

Another important aspect is the NEX process was non-perturbative,evidenced by >95% cell viability after each sampling event Minimalmorphological changes were observed even after sampling every day for 20days from human astrocytes, which are known to react promptly toperturbations. Cell viability is a critical metric for longitudinalstudies. For example, 80% cell viability per sample can take an averageof 3 samples before cell death, while 95% viability gives an average of14 samples. This will be especially important for longitudinalmeasurements of single cells, where cell apoptosis terminates theexperiment.

There are several other key features that make NEX suitable forlongitudinal studies of cell biology. First, the extracted molecules arespatially separated from the cell culture, allowing for simplecollection using a pipette or microfluidic device. This ensures thatfuture analytical technology improvements can be combined with the NEXprocess as the sampling platform. Second, the patterned NS samplingregion allows scalable numbers of cells to be analyzed, whilemaintaining the normal cell-to-cell connectivity and communicationimportant for cell development and differentiation. Third, the NSplatform was compatible with all cell types tested, including cell lines(CHO) and hiPSC-derived cells (cardiomyocytes or neural cells derived in3D cultures).

This sampling technique may therefore be useful for studying dynamiccellular activity or transformations, for example tracking signalingpathways during differentiation of pluripotent stem cell in vitro andcapturing cellular heterogeneity. The throughput could be increased byintegrating microfluidics to sample from a number of independentcellular wells at once, similar to 96-chamber single cell analysisdesigns. Such systems could impact on the understanding of the cellularmechanisms behind cell development, differentiation, and diseasepathology from bulk populations down to single cells.

For example, FIGS. 17a and 17b illustrate examples of apparatuses,similar to that shown in FIG. 1a-1b that are configured to spatiallyresolved sampling. For example, in some of the apparatuses describedherein (e.g., FIG. 17a ), a second surface 1703 (e.g., a removablesample material collectors) may be positioned immediately underneath thenanostraw membrane 1733, so that the molecules that are extracted fromthe cells 1705 through the nanostraws 1707 can be physically orchemically bound to the second sample material collector(s) when thesample material exits the nanostraws 1707 on the bottom of the nanostrawmembrane 1733.

Placing the second surface 1703 (e.g., removable sample materialcollector(s)) in close proximity to the nanostraw membrane 1733 (e.g.,within tens of micrometers) may allow the extracted molecules to bindbefore they have time to diffuse away laterally, so that the twodimensional spatial resolution of the cells on the nanostraws may bepreserved. This configuration may therefore allow the creation of a mapof extracted molecules on the removable sample material collector(s)that corresponds to the positions of the cells on the nanostrawmembrane, and we will be able to tell what cell each of the extractedmolecules came from.

As mentioned above, in any of these variations, the removable samplematerial collectors may be removed or replaced with each repetition ofthe sampling procedure, providing a time course from the sampled cells.For example, the nanostraw membrane 1073 may be placed on the secondremovable sample material collectors (e.g., surface) to captureextracted cellular content (sample material), and then the nanostrawmembrane, on which the same cells are still attached and relativelyundisturbed, may be moved to another removable sample materialcollector(s) to collect sample material at a different time point. Byrepeating this with new removable sample material collectors, samples ofthe content of cells over time with preserved spatial resolution down toor below single cell resolution may be achieved. At any point duringthis time course, a simulation or perturbation of the cells (e.g.,heating, adding a material, etc.) may be added to stimulate or otherwisetest the cell.

This method and apparatus may therefore be able to follow the molecularcontent of individual cells over time in a massively parallel way (e.g.,up to thousands of cells at the time).

As mentioned above, any appropriate removable sample material collectormay be used. For example, the removable sample material collector can bea permeable membrane that the molecules to capture can go into or bindonto, or it can be a solid support for example glass, metal, or plastic.The captured molecules (sample material) can, for example, be physicallyadsorbed, e.g., electrostatically bound in an oppositely charged polymermembrane. For example, mRNA is negative and can be electrostaticallybound in a positively charged polymer membrane. The removable samplematerial collector may be a solid support, and may have capturing agentson it. Depending on what is to be captured (e.g., proteins, metabolites,small molecules), different capture molecules may be bound on theremovable sample material collector and/or different regions of theremovable sample material collector.

For example, for mRNA capture and analysis, poly(dT)s that specificallybind the poly (A) tails of the mRNA may be immobilized on the removablesample material collector or regions of the removable sample materialcollector (e.g., corresponding to specific sample regions). The spatialinformation may be preserved by keying the removable sample materialcollector to match with a unique alignment with the sample regionsand/or nanostraw membrane. Another way to preserve the spatialinformation is to add positional barcodes into the DNA-poly(dT)-capturemolecules. Then mRNA can be converted to cDNA, which can be sequenced bystandard bulk methods. smFISH or similar imaging methods may be used todetect a smaller number of mRNAs with single molecule resolution andwith preserved spatial resolution.

FIG. 17b shows another variation of an apparatus as described herein. Inany of these apparatuses, the bottom electrode 1717 for electroporationcan be placed on the bottom of the nanostraw membrane, as shown in FIG.17b , or, if the removable sample material collector is ionicallyconducting, the bottom electrolyte can extend on both sides of thecapturing removable sample material collector, and the electrode can beplaced underneath the removable sample material collector, as shown inFIG. 17a . In FIG. 17A, the removable sample material collector 1703′ issupported by a second surface 1703. The top electrode 1709 in both casesis above the nanostraw substrate 1723.

Any of the methods (including user interfaces) described herein may beimplemented as software, hardware or firmware, and may be described as anon-transitory computer-readable storage medium storing a set ofinstructions capable of being executed by a processor (e.g., computer,tablet, smartphone, etc.), that when executed by the processor causesthe processor to control perform any of the steps, including but notlimited to: displaying, communicating with the user, analyzing,modifying parameters (including timing, frequency, intensity, etc.),determining, alerting, or the like.

When a feature or element is herein referred to as being “on” anotherfeature or element, it can be directly on the other feature or elementor intervening features and/or elements may also be present. Incontrast, when a feature or element is referred to as being “directlyon” another feature or element, there are no intervening features orelements present. It will also be understood that, when a feature orelement is referred to as being “connected”, “attached” or “coupled” toanother feature or element, it can be directly connected, attached orcoupled to the other feature or element or intervening features orelements may be present. In contrast, when a feature or element isreferred to as being “directly connected”, “directly attached” or“directly coupled” to another feature or element, there are nointervening features or elements present. Although described or shownwith respect to one embodiment, the features and elements so describedor shown can apply to other embodiments. It will also be appreciated bythose of skill in the art that references to a structure or feature thatis disposed “adjacent” another feature may have portions that overlap orunderlie the adjacent feature.

Terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention.For example, as used herein, the singular forms “a”, “an” and “the” areintended to include the plural forms as well, unless the context clearlyindicates otherwise. It will be further understood that the terms“comprises” and/or “comprising,” when used in this specification,specify the presence of stated features, steps, operations, elements,and/or components, but do not preclude the presence or addition of oneor more other features, steps, operations, elements, components, and/orgroups thereof. As used herein, the term “and/or” includes any and allcombinations of one or more of the associated listed items and may beabbreviated as “/”.

Spatially relative terms, such as “under”, “below”, “lower”, “over”,“upper” and the like, may be used herein for ease of description todescribe one element or feature's relationship to another element(s) orfeature(s) as illustrated in the figures. It will be understood that thespatially relative terms are intended to encompass differentorientations of the device in use or operation in addition to theorientation depicted in the figures. For example, if a device in thefigures is inverted, elements described as “under” or “beneath” otherelements or features would then be oriented “over” the other elements orfeatures. Thus, the exemplary term “under” can encompass both anorientation of over and under. The device may be otherwise oriented(rotated 90 degrees or at other orientations) and the spatially relativedescriptors used herein interpreted accordingly. Similarly, the terms“upwardly”, “downwardly”, “vertical”, “horizontal” and the like are usedherein for the purpose of explanation only unless specifically indicatedotherwise.

Although the terms “first” and “second” may be used herein to describevarious features/elements (including steps), these features/elementsshould not be limited by these terms, unless the context indicatesotherwise. These terms may be used to distinguish one feature/elementfrom another feature/element. Thus, a first feature/element discussedbelow could be termed a second feature/element, and similarly, a secondfeature/element discussed below could be termed a first feature/elementwithout departing from the teachings of the present invention.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising” means various components can be co-jointlyemployed in the methods and articles (e.g., compositions and apparatusesincluding device and methods). For example, the term “comprising” willbe understood to imply the inclusion of any stated elements or steps butnot the exclusion of any other elements or steps.

In general, any of the apparatuses and methods described herein shouldbe understood to be inclusive, but all or a sub-set of the componentsand/or steps may alternatively be exclusive, and may be expressed as“consisting of” or alternatively “consisting essentially of” the variouscomponents, steps, sub-components or sub-steps.

As used herein in the specification and claims, including as used in theexamples and unless otherwise expressly specified, all numbers may beread as if prefaced by the word “about” or “approximately,” even if theterm does not expressly appear. The phrase “about” or “approximately”may be used when describing magnitude and/or position to indicate thatthe value and/or position described is within a reasonable expectedrange of values and/or positions. For example, a numeric value may havea value that is +/−0.1% of the stated value (or range of values), +/−1%of the stated value (or range of values), +/−2% of the stated value (orrange of values), +/−5% of the stated value (or range of values), +/−10%of the stated value (or range of values), etc. Any numerical valuesgiven herein should also be understood to include about or approximatelythat value, unless the context indicates otherwise. For example, if thevalue “10” is disclosed, then “about 10” is also disclosed. Anynumerical range recited herein is intended to include all sub-rangessubsumed therein. It is also understood that when a value is disclosedthat “less than or equal to” the value, “greater than or equal to thevalue” and possible ranges between values are also disclosed, asappropriately understood by the skilled artisan. For example, if thevalue “X” is disclosed the “less than or equal to X” as well as “greaterthan or equal to X” (e.g., where X is a numerical value) is alsodisclosed. It is also understood that the throughout the application,data is provided in a number of different formats, and that this data,represents endpoints and starting points, and ranges for any combinationof the data points. For example, if a particular data point “10” and aparticular data point “15” are disclosed, it is understood that greaterthan, greater than or equal to, less than, less than or equal to, andequal to 10 and 15 are considered disclosed as well as between 10 and15. It is also understood that each unit between two particular unitsare also disclosed. For example, if 10 and 15 are disclosed, then 11,12, 13, and 14 are also disclosed.

Although various illustrative embodiments are described above, any of anumber of changes may be made to various embodiments without departingfrom the scope of the invention as described by the claims. For example,the order in which various described method steps are performed mayoften be changed in alternative embodiments, and in other alternativeembodiments one or more method steps may be skipped altogether. Optionalfeatures of various device and system embodiments may be included insome embodiments and not in others. Therefore, the foregoing descriptionis provided primarily for exemplary purposes and should not beinterpreted to limit the scope of the invention as it is set forth inthe claims.

The examples and illustrations included herein show, by way ofillustration and not of limitation, specific embodiments in which thesubject matter may be practiced. As mentioned, other embodiments may beutilized and derived there from, such that structural and logicalsubstitutions and changes may be made without departing from the scopeof this disclosure. Such embodiments of the inventive subject matter maybe referred to herein individually or collectively by the term“invention” merely for convenience and without intending to voluntarilylimit the scope of this application to any single invention or inventiveconcept, if more than one is, in fact, disclosed. Thus, althoughspecific embodiments have been illustrated and described herein, anyarrangement calculated to achieve the same purpose may be substitutedfor the specific embodiments shown. This disclosure is intended to coverany and all adaptations or variations of various embodiments.Combinations of the above embodiments, and other embodiments notspecifically described herein, will be apparent to those of skill in theart upon reviewing the above description.

1. (canceled)
 2. A system for nondestructively sampling intracellularmaterial, the system comprising: a cell culture chamber having an upperregion and a lower region; a nanostraw substrate positioned over thelower region, wherein the substrate comprises a plurality of sampleregions; a plurality of nanostraws extending through the nanostrawsubstrate in each sample region, wherein each nanostraw has an outerdiameter configured to support a cell; a plurality of sample materialcollectors, wherein each sample material collector corresponds to onesample region of the plurality of sample regions; a first electrode inthe upper region; a second electrode in the lower region; and acontroller coupled to the first electrode and the second electrode andconfigured to apply a pulsed voltage through the plurality of nanostrawsof between about 1 V and 100V, a pulse width of between about 10microseconds and 50 milliseconds for a duration of between 1 second and300 seconds.
 3. The system of claim 2, wherein the outer diameter ofeach nanostraw is configured to support a cell without penetrating thecell's cell membrane.
 4. The system of claim 2, wherein the nanostrawsubstrate comprises a pattern of recessed sample regions.
 5. The systemof claim 2, wherein the nanostraw substrate comprises a removablecapture substrate configured to be removably placed into the cellculture chamber.
 6. The system of claim 2, wherein the nanostrawsubstrate is keyed to fit within the cell culture chamber in a uniqueorientation.
 7. The system of claim 2, wherein the nanostraw substratecomprises a polycarbonate membrane.
 8. The system of claim 2, whereinthe nanostraw substrate comprises a blocking coating covering a surfaceof the nanostraw substrate between the sample regions.
 9. The system ofclaim 2, wherein a thickness of samples regions of the nanostrawsubstrate is between 10 nm and 5 microns.
 10. The system of claim 2,wherein the lower region of the cell culture chamber comprises aplurality of sample ports, wherein each sample material collector isassociated with a unique sample port.
 11. The system of claim 2, whereineach nanostraw has an outer diameter between about 20 nm to about 1500nm.
 12. The system of claim 2, wherein each nanostraw has an outerdiameter of greater than 100 nm.
 13. The system of claim 2, wherein theplurality of nanostraws are alumina nanostraws.
 14. The system of claim2, wherein each of the plurality of sample material collectors comprisesa sample material capture substrate configured to bind to the samplematerial.
 15. The system of claim 2, wherein the plurality of samplematerial collectors is removable.
 16. The system of claim 2, wherein thesecond electrode is positioned between the nanostraw substrate and theplurality of sample material collectors.
 17. The system of claim 2,wherein the plurality of sample material collectors is positionedbetween the nanostraw substrate and the second electrode.
 18. The systemof claim 2, wherein the plurality of sampling regions are eachconfigured to have a maximum diameter of between 5 μm and 200 μm. 19.The system of claim 2, further comprising one or more ports into thecell culture chamber configured to automatically switch between cellmedia and media-free buffer, further wherein the controller isconfigured to switch between cell media and media-free buffer beforeapplying the pulsed voltage and to switch between media-free buffer andcell media after applying the pulsed voltage.
 20. The system of claim 2,wherein the controller is configured to sample material from within acell at multiple time points by repeatedly applying a voltage betweenthe upper electrode and the lower electrode through the plurality ofnanostraws, stopping the application of the voltage between the upperand lower electrodes, and allowing the cell to recover for a minimumrecovery time of at least 1 hour before reapplying the voltage.
 21. Asystem for nondestructively sampling intracellular material, the systemcomprising: a cell culture chamber having an upper region and a lowerregion; a nanostraw substrate positioned over the lower region, whereinthe substrate comprises a pattern of recessed sample regions; aplurality of nanostraws extending through the nanostraw substrate ineach sample region, wherein each nanostraw has an outer diameterconfigured to support a cell without penetrating a cell's cell membrane,wherein the outer diameter is between about 20 nm to about 1500 nm; aplurality of removable sample material collectors comprising a samplematerial capture substrate, wherein each sample material collectorcorresponds to one sample region of the plurality of sample regions; afirst electrode in the upper region; a second electrode in the lowerregion; and a controller coupled to the first electrode and the secondelectrode and configured to apply a pulsed voltage through the pluralityof nanostraws of between about 1 V and 100V, a pulse width of betweenabout 10 microseconds and 50 milliseconds for a duration of between 1second and 300 seconds.